The insulin-like growth factor (hereinafter “IGF”) signaling system (hereinafter “IGF axis”) is comprised of the ligands IGF-I, IGF-II and insulin, and a family of transmembrane receptors including the insulin type 1- and type 2-IGF receptors (Daughaday and Rotwein, Endocr. Rev. 10:68-9, 1989; Werner et al., Insulin-like growth factors: Molecular and Cellular Aspects, CRC Press, LeRoith (eds.), Boca Raton, pp. 17-47, 1991.
The IGF axis also includes the insulin-like growth factor binding proteins (hereinafter “IGFBPs”). Six IGFBPs have been identified, cloned and sequenced (Baxter & Martin, Prog. Growth Factor Res. 1:49-68, 1989; Rosenfeld et al., Recent Progress Hormone Res. 46:99-159, 1991; Shimasaki & Ling, Prog. Growth Factor Res. 3:243-266, 1991). They share a high degree of similarity in their primary protein structure, particularly in the corresponding N- and C-terminal regions (58% and 34% similarity, respectively), which are separated by a variable mid-protein segment of 55 to 95 amino acid residues (Shimasaki & Ling, supra). The IGFBPs bind IGF-I and IGF-II, but not insulin, with high affinity (Jones & Clemmons, Endocr. Rev. 16:3-34, 1995). The IGFBPs appear to serve essential functions of transporting the IGFs, prolonging IGF half-lives, and regulating the availability of free IGFs for interaction with IGF receptors.
Thus, the IGFBPs modulate the effects of IGFs on growth and differentiation (Jones & Clemmons, supra; Lowe, Insulin-like growth factors: Molecular and Cellular Aspects, CRC Press, LeRoith (eds.), Boca Raton, pp. 49-85, 1991; Oh et al., Growth Regul. 3: 113-123, 1993; Kelley et al., Int. J. Biochem. Cell Biol. 28:619-637, 1993; Rajaram et al., Endocr. Rev. 18:801-831, 1997). Some IGFBPs (e.g., IGFBP-3) may also act as growth-suppressing factors in various cell systems through IGF-independent mechanisms (Oh, Endocrine. 7:111-113, 1997; Oh, Breast Cancer Res. Treat. 47:283-293, 1998).
Additional potential IGF binding proteins, referred to as IGFBP related proteins (hereinafter “IGFBP-rPs”), have been recently identified that have a significant similarity to the IGFBPs in their N-terminal domains (Hwa et al., Acta Ped. Scand. 428:37-45, 1999). Collectively, current data supports the broad concept of an “IGFBP superfamily” with both high- and low-affinity members, wherein at least some members influence cell growth and differentiation by both IGF-dependent and IGF-independent means (Hwa et al., supra; Baxter et al., Endocrinology 139:4036, 1998; Baxter et al., J. Clin. Endocrinol. Metab. 83:3213, 1998).
The human IGFBP superfamily is currently comprised of six high-affinity species (IGFBPs 1-6), and nine low-affinity IGFBP-related proteins (IGFBP-rPs). Structural characteristics of various members of the human IGFBP superfamily are summarized in Table 1.
TABLE IIStructural Characteristics of the Human IGFBP SuperfamilyMolecularNumber ofNumber ofN-linkedChromosomalmRNA sizeIGFBPWeightamino acidscysteinesglycosylationlocalization(kb)High affinity IGFBP related proteinsIGFBP-125,27123418No7p1.6IGFBP-231,35528918No2q1.5IGFBP-328,71726418Yes7p2.4IGFBP-425,95723720Yes17q1.7IGFBP-528,55325218No2q1.7, 6.0IGFBP-622,84721616No12 1.1Low affinity IGFBP related proteinsIGFBP-rP1?25118Yes4q1.1IGFBP-rP2?349 (pre)39Yes6q2.4IGFBP-rP3?357 (pre)41? (No)8q2.4IGFBP-rP4?379 (pre)35? (No)?2.4
IGFBP-3 is the principal IGFBP in adult serum, where it circulates as a 150 kDa-complex consisting of IGFBP-3, an acid-labile subunit, and IGF peptide (Neely et al., Acta Pediatr. Scand. 372:116-123, 1991; Oh et al., Growth Regul. 3:113-123, 1993; Ruoslahti & Pierschbacher, Science 238:491-493, 1987). Its principal role has been postulated to be transporting IGFs and protecting them from rapid clearance and/or degradation (Hintz & Liu, J. Clin. Endocrinol. Metab. 45:988-982, 1977; Baxter et. al., Biochem. Biophys. Res. Commun. 139:1256-1259, 1986; Baxter & Martin, Prog. Growth Factor Res. 1:49-56, 1989).
The IGF Axis in the Human Mammary System
IGF-I, IGF-II, type-1 and type-2 IGF receptors, and the IGFBPs. The IGFs are major regulators of mammary epithelial and breast cancer cell growth (Jones & Clemmons, Endocr. Rev. 16:3-34, 1995; Oh, Endocrine. 7:111-113, 1997; Oh, Breast Cancer Res. Treat. 47:283-293, 1998; De Leon et al., Growth Factors 6:327-336, 1992; Furlanetto & DiCarlo, Cancer Res. 44:2122-2128, 1984; Huff et al., Cancer Res. 46:4613-4619, 1986). IGF-I and IGF-II, for example are potent mitogens for a number of breast cancer cell lines in vitro (De Leon et al., supra; Huff et al., supra; Westley & May, Reviews on Endocrine-Related Cancer 39:29-34, 1991; Baxter et al., Insulin-like Growth Factors/Somatomedins, De Gruyter, Spencer (ed.), Berlin, pp 615-618, 1983). Moreover, IGF-I and IGF-II mRNAs are detectable in the majority of human breast tumor specimens (Cullen et al., Cancer Res. 51:4978-4985, 1991; Paik, Breast Cancer Res. Treat. 22:31-38, 1992). Virtually all breast tumor specimens, and cell lines derived therefrom, express and produce type-1 and type-2 IGF receptors, and insulin receptors (Cullen et. al., Cancer Res. 50:48-53, 1990; Bonneterre et al., Cancer Res. 50:6931-6935, 1990; Peyrat & Bonneterre, Breast Cancer Res. Treat. 22:59-67, 1992; De Leon et. al., Biochem. Biophys. Res. Commun. 152:398-405, 1988). The mitogenic effects of both IGF-I and IGF-II are mediated by the type-1 IGF receptor, as determined through the use of estrogen-dependent breast cancer cells (De Leon et al., Growth Factors 6:327-336, 1992; Papa et. al., Mol. Endocrinol. 5:709-717, 1991).
In contrast, relatively little is known about the molecular mechanisms and biological functions of the IGFBPs in the context of breast cancer. Specifically, breast cancer cells are known to secrete various types of IGFBPs, and these appear to regulate the availability of free IGFs for interaction with IGF receptors (Lamson et al., Growth Factors 5:19-28, 1991). The predominant secreted IGFBP appears to correlate with the estrogen receptor status of the cell (Figueroa et al., J. Cell. Biochem. 52:196-205, 1993). Estrogen-nonresponsive (estrogen receptor (ER)-negative) cells secrete predominantly IGFBP-3 and IGFBP-4 as major species, and IGFBP-6 as a minor one. Estrogen-responsive (ER-positive) cells secrete IGFBP-2 and IGFBP-4 as major species, and IGFBP-3 and IGFBP-5 as minor ones.
Therefore, the IGF axis in breast cancer is complex, involving autocrine, paracrine, or endocrine-derived IGFs that bind to specific cellular receptors and thereby elicit, inter alia, IGFBP secretion by the target cells. The IGFBPs, in turn, appear to regulate the availability of free IGFs for interaction with IGF receptors. However, the broader biological significance of IGFBPs generally, or in the particular context of breast cancer is unclear. Moreover, the basis and significance of variations in IGFBP secretory specificity are unknown, and the predominant species may vary significantly depending on the estrogen responsivess of the secreting cells.
IGFBP-3 and its anti-proliferative action in human breast cancer cells. Expression of IGFBP-3 in human breast cancer cells is hormonally regulated, and IGFBP-3 is known to inhibit cancer cell growth through both (a) IGF-dependent, and (b) IGF-independent mechanisms.
(a) IGF-dependent anti-proliferative action. IGFBP-3 is known to indirectly inhibit cancer cell growth through IGF-dependent interactions. IGFBP-3 is the predominant IGF-binding protein in human serum where it circulates as part of a 150 kDa ternary complex. The binding affinity of IGFBP-3 for IGF peptides is generally higher than that of the type I and type II IGF cell-surface receptors, implying that IGFBP-3 can modulate IGF binding to its receptor, thereby blocking local IGF-dependent biological action. In fact, co-incubation of cells with IGFBP-3 and IGF peptides results in an inhibition of the IGF-dependent mitogenic effect in human breast cancer cells, in vitro (Martin et al., Endocrinology 136:1219-1226, 1995). In situ hybridization studies of the expression of IGF-I and IGF-II in human breast cancer tissues indicate that IGF-I mRNA is detected only in stromal cells, and not in normal or malignant epithelial cells implying that IGF-I may function as a paracrine stimulator of epithelial cells. In contrast, IGF-II mRNA was expressed in both malignant epithelial cells and their adjacent stromal cells (Paik, Breast Cancer Res. Treat. 22:31-38, 1992). Both paracrine or autocrine effects of IGF peptides can be modulated in vivo by IGFBP-3 produced by epithelial cells.
This IGF-dependent mechanism for IGFBP-3 inhibition of cancer cell growth is consistent with IGFBP-3 proteolysis studies. Post-translationally, IGFBP-3 can be proteolyzed by proteases such as cathepsin D, prostate-specific antigen (PSA) and plasmin, that are detectable in human breast cancer cells (Capony et. al., Cancer Res. 49:3904-3909, 1989; Conover & De Leon, J. Biol. Chem. 269:7076-7080, 1994; Yuet et al., Clinical Biochem. 27:75-79, 1994; Cohen et al., J. Clin. Endocrinol. Metab. 75:1046-1053, 1993; Schmitt et al., Biomedica Biochemica Acta 50:731-741, 1991; Lee et al., J. Clin. Endocrinol. Metab. 79: 1376-1382, 1994). In general, IGFBP-3 proteases are postulated to lower the affinity of IGFBP-3 for IGF, thereby increasing the availability of IGFs to cell-membrane receptors. PSA, for example, has been shown to reverse the inhibitory effect of IGFBP-3 on IGF-stimulated prostate cell growth by cleaving IGFBP-3 and generating IGFBP-3 fragments with lower affinity for IGFs (Cohen et al., J. Clin. Endocrinol. Metab. 73:401-407, 1991). However, the broad biological significance and molecular actions of IGFBP-3 proteolysis are unclear in the context of human breast cancer.
(b) IGF-independent anti-proliferative action. IGFBPs may also have specific IGF-independent biological effects in various cell systems, including human breast cancer cells. For example, exogenously added IGFBP-3 (in the absence of added IGF) inhibits estrogen-stimulated breast cancer cell proliferation (Pratt & Pollak, Biochem. Biophys. Res. Commun. 198:292-297, 1994). Moreover, estrogen can inhibit expression and secretion of IGFBP-3, whereas anti-estrogens (e.g., tamoxifen and ICI 182,780) stimulate production of IGFBP-3 in ER-positive human breast cancer cells.
Likewise, the expression and production of IGFBP-3 is specifically stimulated by TGF-β and retinoic acid (“RA”) in human breast cancer cells, consistent with a possible role for IGFBP-3 in TGF-β- and RA-induced growth inhibition (Martin et al., Endocrinology 136:1219-1226, 1995; Fontana et al., Endocrinology 128:1115-1122, 1991; Oh et al., J. Biol. Chem. 270:13589-13592, 1995). The anti-proliferative effects of TGF-β, RA, TNF-α and vitamin-D analogs in human breast cancer cells appear to be partially mediated through the IGFBP-3 axis (Oh et al., supra; Gucev et al., Cancer Research, 56:1545-50, 1996; Rozen et al., Int. J. Oncol. 13: 865-869, 1998; Colston et al., J. Mol. Endocrinol. 20: 157-162, 1998).
Other studies also support the function of IGFBP-3 as a major IGF-independent growth-suppressing factor in various cell systems. See Valentinis et al., Mol. Endocrinol. 9: 361-367, 1995 (showing that growth rate is significantly reduced in IGFBP-3-transfected fibroblasts with a targeted disruption of the IGF-I receptor gene); Delbe et al., Biochem. Biophys. Res. Commun. 179: 495-501, 1991 (showing that purified mouse IGFBP-3 binds to the chick embryo fibroblast cell surface and inhibits cell growth); Andreatta-Van Leyen et al., J. Cell Physiol. 160: 265-274, 1994 (showing that up-regulation of IGFBP-3 expression is correlated with RA-induced cell growth inhibition in cervical epithelial cells); and Buckbinder et al., Nature 377: 646-649, 1995 (showing that the tumor suppressor p53 induces IGFBP-3 expression, indicating that IGFBP-3 may be a mediator in p53 signaling).
Additional studies support the ability of IGFBP-3 to induce apoptosis. For example, in MCF-7 cells, the treatment with recombinant human IGFBP-3 for 72 hours has been shown to increase apoptosis and to inhibit [3H]-thymidine incorporation (Nickerson et al., Biochem. Biophys. Res. Commun. 237:690-693, 1997). In Hs578T human breast cancer cells, IGFBP-3 results in no direct induction of apoptosis, but preincubation of the cells with IGFBP-3 caused a dose-dependent potentiation of apoptosis by ceramide, an apoptosis-inducing agent, consistent with an IGF-independent activity of IGFBP-3 (Gill et al., J. Biol. Chem. 272:25602-25606, 1997).
Therefore, IGFBP-3 is an important IGF-independent anti-proliferative factor for human breast cancer cells.
The IGF Axis in Human Chondrocyte Proliferation and Differentiation
Endochondral bone development results from the condensation and proliferation of mesenchymal cells, which in turn, mature into differentiated and terminally differentiated chondrocytes, forming cartilaginous templates that are subsequently replaced by bone. During the process of chondrocyte proliferation and differentiation (“chondrogenesis”), chondrocytic cells progress through an ordered program of proliferation and differentiation. This program is strictly regulated, so that proper bone length is maintained. Several hormones and growth factors have been demonstrated to regulate differentiation rate through antiproliferative and/or apoptotic mechanisms (Serra et al., J. Cell. Biol. 139:541-552, 1997; Deng et al., Cell 84:911-921, 1996; Kronenberg et al., Recent Prog. Horm. Res. 53:283-303, 1998).
Insulin-like growth factors (IGFs) have been shown to play a central role in chondrogenesis (Spagnoli & Rosenfeld Endocrinol Metab. Clin. North Am. 25:615-631, 1996). IGFBP-3 sequesters IGF, inhibiting binding to the IGF-I receptor, and in some cell systems also has a direct (i.e., IGF-independent) effect on cell replication. However, the functional relationship between IGF and IGFBP-3 in endochondral bone growth is not well understood.
IGFBP-3 Receptor (“P4.33”)
Finally, it is known that IGFBP-3 can bind to the cell surface and act as a growth inhibitor for ER-negative human breast cancer cells (Oh et al., J. Biol. Chem. 270:13589-13592, 1995). The interaction of IGFBP-3 with the breast cancer cell surface and its subsequent biological effects appear to involve IGFBP-3-specific cell surface association proteins that may mediate the direct inhibitory effect of IGFBP-3 on the growth of cells in monolayer (Oh et al., J. Biol. Chem. 268:26045-26048, 1993).
The present applicants previously identified and characterized a novel protein, P4.33, that interacts specifically with IGFBP-3 in an IGF-independent manner (see International Patent Application PCT/US01/16437, incorporated by reference herein in its entirety). P4.33 functions as a receptor for IGFBP-3, and is thereby involved in the inhibition of DNA synthesis and cellular proliferation, and in the induction of apoptosis in cancer cells.
However, because IGFBP-3 acts in vivo and in vitro as a bivalent molecule, binding to both IGF and to P4.33 (i.e., modulates cancer cell growth and apoptosis via both IGF-dependent and independent mechanisms), assessing the physiological properties of potential therapeutic compounds is complex and troublesome. A modulator (e.g., antagonist) of the IGFBP-3/P4.33 interaction might act indirectly (i.e., allosterically) through interaction at the IGF binding site of IGFBP-3, rather than by directly antagonizing the P4.33 ligand-binding site. Thus, screening assays based on modulating the IGFBP-3/P4.33 binding interaction would identify, in addition to those compounds that affect IGF-independent processes (because they directly interact with IGFBP-3/P4.33 complex), compounds that affect IGF-dependent processes via interaction with the IGF binding site of IGFBP-3.
Therefore, there is a need in the art for effective screening assays for the identification of specific antagonists and agonists of P4.33 (i.e. to identify specific antagonist and agonists of IGF-independent processes). There is a need in the art to identify novel methods and compositions for the treatment of cancer, and cartilage and bone disorders. There is a need in the art to further understand the role of IGFBP-3 in condrogenesis and bone growth. There is a need in the art to identify novel IGFBP-related polypeptides that have no, or diminished binding to IGFs, but retain affinity for the IGFBP-3 receptor.